This invention relates generally to derivatized fullerenes useful for therapeutic and in vivo diagnostic applications which exhibit improved biodistribution. In particular, the invention relates to methods of making and using such derivatives and more particularly to the use of such derivatives for improved magnetic resonance imaging (MRI).
The use of fullerenes as diagnostic and therapeutic compounds has been discussed in the open literature since at least 1991. A suggestion for the use of C60 in cancer diagnosis and therapy even appeared in Popular Science Magazine (Edelson 1991). U.S. Pat. No. 5,994,410 (Long et al.) relates to therapeutic use of certain water-soluble fullerene derivatives F—(X)m where F is the fullerene core, m is 2-40, X is OH, (CH2)n-SO3H, or a metal salt of (CH2)n-SO3− and n is 2-50 for treating a free radical-related medical condition.
U.S. Pat. No. 5,688,486 issued Nov. 18, 1997 by Watson et al. based upon a PCT WO/93/15768, published Aug. 19, 1993 relates to the use of fullerenes and metallofullerenes for diagnostic and therapeutic applications. The patent provides a number of examples of fullerenes that are purported to be useful as carriers for diagnostic or therapeutic agents. Examples include C60Fn′ where n′ is 30 to 60 for use as a PET contrast agent; Mtn@Cm (Mt=lanthanide, transition or rare earth metal, m=60, 80, 82, 84, 92, 106 etc., and n=1, 2, etc.) with paramagnetic cage complexes useful as MRI contrast agents and certain other metal cage complexes (lanthanum, iridium and lutetium) useful in photodynamic therapy; polyiodinated C60 useful as an X-ray contrast agent; C60(CH2C6H5)n′ n′=3,5 radical useful as MRI contrast agents; polyhydroxylated Gd@C60 prepared by the method of Chiang et al., 1992; Gd@C82 reported to be derivatizable by the method of Chiang et al., 1992; Gd@C60 imbedded in cyclodextrin for use in MR imaging (also M@C60 where M is Dy, Ho, La, Lu, and other rare earth metals); osmylated fullerene for tissue staining; {[(C2H5)3P]2Pt}6C60 for use as an X-ray contrast medium; sugar-labeled fullerenes for enzyme assays; (eta-5-C9H7)Ir(CO)(C60) for photodynamic therapy for cancer; isotopically-labeled fullerenes (carbon-14 enriched C60) as a source of countable radiation in a diagnostic test; fluorinated fullerenes for use in NMR imaging; C60 species labeled with carborane units for use in anti-cancer therapy using neutron irradiation; and 111In@C80 useful as a radiation source for SPECT imaging; C60Br24 for use in CT scanning. The patent notes the use of fullerene materials as contrast-enhancing agents in MRI, ultrasound, PET, Overhauser MRI, scintigraphy, X-ray, CT, SPECT, magnetometric tomography, EIT, visible and it imaging and as carriers for signal reporters, such as chromophores, fluorophores or radiolabels as well as in in vitro assays and for tissue staining. The patent further notes therapeutic applications of fullerene materials to carry and release therapeutically active molecules or atoms or in photodynamic therapy or radiotherapy or as therapeutically active bioconjugates.
U.S. Pat. No. 6,265,443 reports a method for treatment of neurotoxic injury resulting form the release of oxygen-derived free radicals using carboxylated derivatives of C60(C(COOH)2)n, where n is 1-4.
U.S. Pat. Nos. 5,811,460 and 6,204,391 report water soluble derivatives of C60 for inactivation of HIV. The derivatives a re generally described as symmetrically substituted with organic moieties comprising from 1 to about 20 carbon atoms each and optionally comprising polar heteroatoms, such as oxygen and nitrogen. The patents illustrate the structures of a number exemplary derivatized C60 molecules with substituent groups added at one or two of the double bonds of the fullerene. An exemplary derivatized C60 is designated 4,4′-bis(HOC(O)(CH2)2C(O)NH(CH2)2-)diphenyl-C61.
U.S. Pat. Nos. 6,162,926 and 6,399,785 relate to multiply-substituted fullerene derivatives and to methods of producing a large number of multiply-substituted fullerene derivatives to generate combinatorial libraries wherein some of the compounds of the library are purported to possess pharmaceutical, materials science, or other utilities. The patent provides methods and lists references providing methods for derivatization of fullerenes, for example, via various cycloaddition reactions (1,3-dipolar additions, Diels-Alder reactions, etc.), cyclopropanation by addition/elimination and by addition of carbanions, alkyl lithium reagents or Grignard reagents. These patents are incorporated by reference herein specifically for the derivatization methods described and referenced therein and for structures of derivatized fullerenes that are illustrated therein.
U.S. Pat. No. 6,355,225 relates to the use of water-soluble, air-stable paramagnetic fullerenes having an unpaired electron useful as contrast agents for MRI imaging and spectroscopy. The fullerenes of the invention are exemplified by fullerols and particularly by radicals of C60(OH)x (where x is 12 or 32). The patent reports a relaxivity (r1) measurement of 0.5 mM−1 sec−1 for the −1 or −2 anion of C60(OH)32.
Several different groups have identified water-solubilized polyhydroxyl Gd metallofullerene compounds as potential MRI contrast agents Zhang et al. 1997; Wilson et al., 1999; Mikawa, et al., 2001.)
U.S. Pat. No. 6,471,942 relates to the use of trimetallic nitride endohedral metallofullerenes having at least one diagnostic atom and at least one treatment atom encapsulated within a fullerene cage for imaging and treating an area of the body. The patent indicates generally that the fullerene of the patent may be “modified to enhance absorption” in the body and in target tissues by attaching at least one functional group to the fullerene cage. Functional groups selected from “an aminosubstituted group, a carboxyl group, a hydroxyl group, a polyethylene glycol complex, carbohydrates, amino acids, proteins, nucleic acids, markers and antibodies.”
While the general idea for the use of fullerene and metallofullerene compounds having utility in medicine and diagnostics has been discussed in the prior art, with emphasis on MRI applications, few of the compounds synthesized and indicated to be useful for such applications to date have significant utility in general for such applications because they are not sufficiently water-soluble.
In addition to being extremely hydrophobic, metallofullerene molecules have a strong propensity to polymerize and/or to aggregate when in water. The hydroxyl groups suggested in the prior art for water solubilization do not prevent aggregation, as a result the polyhydroxylated metallofullerenes are nano-aggregates that range in size from 10-100 nm or larger. When introduced in-vivo, the body's reticuloendothelial system recognizes that these compounds are actually small particles, not individually solvated molecules. They are subsequently encapsulated by phagocytosis and carried to the RES tissues (liver, spleen, bone marrow and lymph nodes). This pharmacokinetic (PK) behavior is unsuitable for broad use in medical imaging. This PK behavior is also not desirable for general MRI contrast agents, although it may be acceptable for RES contrast agents.
Biodistribution studies of the polyhydroxylated metallofullerenes have recently revealed high uptake levels of these compounds by the reticuloendothelial system (RES). A radiotracer study conducted by Cagle et al., 1999 with 166Hon@C82(OH)x (n=1, 2; x˜16) showed significant RES uptake in mice, including concentration of the polyol in liver and bone. An MR imaging and biodistribution study by Mikawa et al., 2001 with Gd@C82(OH)x (x˜40) reported similar results. A radiotracer study with the (reportedly non-endohedral) polyhydroxyl C60 derivative 99mTc—C60(OH)x in mice and rabbits also showed significant uptake of the polyhydroxyl fullerene by the kidneys, bone, spleen and liver (Qingnuan et al, 2002.) In spite of the reported high r1 values for the polyhdroxylated metallofullerenes, these biodistribution studies indicate that these fullerenes (e.g., Gd@C82(OH)x, and Ho@C82(OH)), will only have limited use as MRI contrast agents, i.e. for imaging the reticuloendothelial system (liver, spleen, bone marrow)
Two reports on in vivo absorption, distribution and excretion of fullerenes are consistent with the recent biodistribution results reported for polyhydroxylated metallofullerenes. Yamago et al., 1995 relates to studies using two water-soluble mono-derivatized C60 compounds 1 and 2:
Compound 1 (radioactively labeled with 14C) was reported to not be effectively absorbed when administered orally, but the small amount absorbed moved quickly to the liver and to other tissues and thereafter excretion was slow with over 90% of what was absorbed retained in the body after one week. When delivered intravenously, 73% of radioactively labeled compound 1 was found in the liver after 1 h and 80% of the radioactivity was retained in the liver after 30 h. From 30-160 h the radioactivity in the various organs decreased but was distributed in skeletal muscle and hair, without excretion from the body. Compound 2 was found to exhibit no acute toxicity. Although fullerene 2 did not exhibit acute toxicity, the authors state that the administered fullerene was retained in the body for long periods which “raise new concerns about chronic toxicity.”
Bullard-Dillard et al., 1996 reported that C60 intravenously injected into rats as a fine suspension in water and a more water soluble quaternary ammonium salt derivatized C60 injected as an ethanol-containing solution both predominantly accumulated (90-95% and 52%, respectively) in the liver. Further C60 was reported not to be eliminated from the liver over the 120-h period of the study. The authors state, based on their results, that while C60 is not acutely toxic its use in vivo would likely lead to long-term accumulation in the liver.
To realize the full potential of the fullerene and metallofullerene compounds in medicine and diagnostics, other derivatives that are eliminated through the kidneys in a shorter time frame and which do not accumulate in the RES tissues are needed.